HER2-positive breast cancer (HER2+ BC) is an aggressive subtype with a poor prognosis. Although the monoclonal antibody Herceptin, which targets the HER2 receptor, has improved survival rates in patients with HER2+ BC, many patients present with de novo Herceptin resistance. Furthermore, the majority of patients that initially respond to Herceptin treatment will ultimately develop resistance. To overcome the devastating effects of de novo and acquired Herceptin resistance, new therapeutic targets must be identified. The purpose of this proposal is to define how the novel oncogene FAM83A (for FAMily with sequence similarity 83, member A), drives Herceptin resistance and to determine how FAM83A might be targeted to overcome resistance. FAM83A is overexpressed in HER2+ BCs, particularly Herceptin-resistant cells. In fact, selection for Herceptin resistance results ina significant increase in FAM83A expression. Importantly, inhibition of FAM83A represses Herceptin-resistant cell proliferation, turns off downstream effector signaling, and re-sensitizes cells to Herceptin. Taken together, our preliminary studies implicate FAM83A is key signaling component in Herceptin resistant cells and provide a proof-of-principle that targeting FAM83A may have clinical benefit. The short-term goal of our proposed studies is to provide a greater understanding of the molecular mechanism by which FAM83A promotes Herceptin-resistant HER2 signaling. The results generated from the proposed studies will be critical for our long-term goal of therapeutically targeting FAM83A-mediated signaling complex formation. To define the role of FAM83A in Herceptin resistance, two specific aims are proposed. In Aim 1, we will determine how elevated FAM83A contributes to hyperactive receptor-activated signaling in Herceptin resistant BC. By experimentally manipulating FAM83A levels in a panel of Herceptin-sensitive and Herceptin-resistant cancer cells in vitro and patient-derived tumors in vivo, we propose to define how elevated levels of this novel oncogene alter the activation status of HER2, downstream effector signaling, and HER2-depoendent gene expression signatures. In Aim 2, we will identify key proteins that FAM83A brings to the HER2 receptor to promote Herceptin-resistant HER2 signaling and begin to interrogate their role in Herceptin resistance. Completion of the proposed studies will provide a clearer understanding of how FAM83A regulates the complex signaling cascades that mediate Herceptin resistance. Defining the role of FAM83A in modulating the signaling complexes that promote Herceptin resistance will provide a greater understanding of HER2-driven tumorigenesis and targeted therapy resistance. The insight gained from our proposed studies will lay the foundation for targeting FAM83A-mediated signaling hyperactivation, bringing us closer to overcoming the devastating effects of Herceptin resistance.